This invention relates to novel vitamin D analogues which show strong activity in inducing differentiation and inhibiting undesirable proliferation of certain cells, including skin cells and cancer cells, as well as immunomodulating effects, to pharmaceutical preparations containing these compounds, to dosage units of such preparations, and to their use in the treatment and prophylaxis of diseases characterized by abnormal cell differentiation and/or cell proliferation.
It has been shown that 1xcex1,25-dihydroxy-vitamin D3 (1,25(OH)2D3) influences the effects and/or production of interleukins (Muller, K. et al., Immunol. Lett., 17 361-366 (1988)), indicating the potential use of this compound in the treatment of diseases characterized by a dysfunction of the immune system, e.g. autoimmune diseases, AIDS, host versus graft reactions, and rejection of transplants or other conditions characterized by an abnormal interleukin-1 production, e.g. inflammatory diseases such as rheumatoid arthritis and asthma.
It has also been shown that 1,25(OH)2D3 is able to stimulate the differentiation of cells and inhibit excessive cell proliferation (Abe, E. et al., Proc. Natl. Acad. Sci., U.S.A., 78, 4990-4994 (1981)), and it has been suggested that this compound might be useful in the treatment of diseases characterized by abnormal cell proliferation and/or cell differentiation such as leukemia, myelofibrosis and psoriasis.
Also, the use of 1,25(OH)2D3, or its pro-drug 1xcex1-OH-D3, for the treatment of hypertension (Lind, L. et al., Acta Med. Scand., 222, 423-427 (1987)) and diabetes mellitus (Inomata, S. et al., Bone Mineral., 1, 187-192 (1986)) has been suggested. Another indication for 1,25(OH)2D3 is suggested by the recent observation of an association between hereditary vitamin D resistance and alopecia: treatment with 1,25(OH)2D3 may promote hair growth (Editorial, Lancet, March 4, p. 478 (1989)). Also, the fact that topical application of 1,25(OH)2D3 reduces the size of sebaceous glands in the ears of male Syrian hamsters suggests that this compound might be useful for the treatment of acne (Malloy V. L. et al., The Tricontinental Meeting for Investigative Dermatology, Washington, (1989)).
However, the therapeutic possibilities in such indications are severely limited by the well known potent effect of 1,25(OH)2D3 on calcium metabolism; elevated blood concentrations will rapidly give rise to hypercalcemia. Thus, this compound and some of its potent synthetic analogues are not satisfactory for use as drugs in the treatment of e.g. psoriasis, leukemia or immune diseases which may require continuous administration of the drug in relatively high doses.
A number of vitamin D analogues have recently been described that show some degree of selectivity in favour of the cell differentiation inducing/cell proliferation inhibiting activity in vitro as compared with the effects on calcium metabolism in vivo (as measured in increased serum calcium concentration and/or increased urinary calcium excretion), which adversely limit the dosage that can safety be administered. One of the first of these to appear, calcipotriol (INN) or calcipotriene (USAN), has been developed on the basis of this selectivity and is now recognized worldwide as an effective and safe drug for the topical treatment of psoriasis.
A study with another vitamin D analogue, Seocalcitol [1(S), 3(R)-dihydroxy20(R)-(5xe2x80x2-ethyl-5xe2x80x2-hydroxy-hepta-1 xe2x80x2(E), 3 xe2x80x2(E)-diene-1xe2x80x2-yl)-9,10-secopregna-5(Z), 7(E), 10(19)-triene], selected on this basis supports the concept that systemically administered vitamin D analogues may inhibit breast cancer cell proliferation in vivo at sub-toxic doses (Colston, K. W. et al., Biochem. Pharmacol. 44, 2273-2280 (1992) and Mathiasen, I. S. et al., J. Steroid Biochem. Molec. Biol., 46, 365-371 (1993)).
Related compounds having the following formula are disclosed in WO 98/47866: 
wherein A is a single or double bond, T is CH2 or CH2CH2; B is CH2CH2, CHxe2x95x90CH or Cxe2x89xa1C, R1 and R3 are H or OH, C(R,R) is CH2 or Cxe2x95x90CH2, R2 is CH3 and R4 is H, or R2 is H and R4 is CH3, L is phenyl and R5 is OH or C(C1-4-alkyl)2OH, or Lxe2x80x94R5 is 2-furyl which is 5-substituted by C(C1-4-alkyl)2OH, with the proviso that when L is phenyl, A is a single bond, B is Cxe2x89xa1C, T is CH2, each R1 and R3 are OH, C(R,R) is Cxe2x95x90CH2, R2 is CH3, R4 is H, and R5 is C)CH3)2OH, then R5 must be in position ortho or para. These compounds are described in WO 98/47866 as useful in the treatment or prevention of vitamin D dependent disorders, particularly psoriasis, basal cell carcinomas, disorders of keratinization and keratosis, leukemia, osteoporosis, hyperparathyroidism accompanying renal failure, transplant rejection and graft vs. host disease.
The stereochemistry of the compounds described in WO 98/47866 is not given at the double bond (position 22) when B is CHxe2x95x90CH. However, from the exemplification and the syntheses which are disclosed, it is evident that only 22-trans compounds are envisaged.
Notwithstanding the extensive prior art efforts to provide therapeutically effective products, there is a continuing need for new vitamin D analogues with an acceptable combination of desired therapeutic activity and minimum toxic effects. The compounds of the present invention provide hitherto undisclosed vitamin D analogues with cell proliferation inhibitory and cell differentiating promoting activities without the undesired side effects of increased serum calcium levels and skin irritation.
The present invention relates to compounds of the general formula I 
wherein X represents hydrogen or hydroxy; R1 and R2, which may be the same or different, represent hydrogen, (C1-C4)alkyl optionally substituted with one hydroxyl group or one or more fluorine atoms, or, together with the carbon atom to which they are attached, R1 and R2 form a (C3-C5)carbocyclic ring. R3 represents hydrogen, (C1-C4)alkyl, (C1-C4)alkoxy, or a halogen atom, such as fluorine, chlorine, bromine, or iodine.
The configuration of the carbon atoms marked with an asterisk may be both S or R.
The present invention also relates to in vivo hydrolyzable esters of the compounds of general formula I with pharmaceutically acceptable acids.
As will be evident, the compounds of formula I have the cis-configuration at the double bond at position 22. According to the invention, it has been found that the 22-cis compounds are much more active in stimulating cell differentiation and inhibiting undesirable cell proliferation.
In compounds of the invention it is preferred that X represents hydroxy; R1 and R2 are the same and preferably represent (C1-C2)alkyl optionally substituted with one hydroxyl group or one or more fluorine atoms; and R3 represents hydrogen,(C1-C2)alkyl, fluorine or chlorine. Preferred positions of the C(R1)(R2)(X) group are meta and para. Even more preferred are compounds of formula I wherein R1 and R2 both represent methyl, trifluoromethyl, or ethyl, and R3 represents hydrogen.
The invention also includes diastereoisomers of the compounds of formula I in pure form or as a mixture of diastereoisomers of a compound of formula I.
Preferred compounds of the invention are selected from the group consisting of:
1(S), 3(R)-Dihydroxy-20(R)-[2(Z)-(3-(2-hydroxy-2-propyl)-phenyl)-vinyl]-9, 10-secopregna-5(Z), 7(E), 10(19)-triene.
1(S), 3(R)-Dihydroxy-20(S)-[2(Z)-(4-(2-hydroxy-2-propyl)-phenyl)-vinyl]-9,10-secopregna-5(Z), 7(E), 10(19)-triene.
1(S), 3(R)-Dihydroxy-20(R)-[2(Z)-(4-(2-hydroxy-2-propyl)-phenyl)-vinyl]-9,10-secopregna-5(Z), 7(E), 10(19)-triene,
and in-vivo hydrolyzable esters thereof with pharmaceutically acceptable acids.
The term xe2x80x9calkylxe2x80x9d as used herein refers to any univalent group derived from an alkane by removal of a hydrogen atom from any carbon atom, and includes the subclasses of normal alkyl (n-alkyl), and primary, secondary and tertiary alkyl groups respectively, and having the number of carbon atoms specified, including for example methyl, ethyl, n-propyl, i-propyl, n-butyl, sec. butyl, tert. butyl and isobutyl. Alkane refers to an acyclic branched or unbranched hydrocarbon having the general formula CnH2n+2, and therefore consisting entirely of hydrogen atoms and saturated carbon atoms.
xe2x80x9cHalogenxe2x80x9d is intended to indicate fluoro, chloro, bromo or iodo; fluoro, chloro, and bromo being preferred
The Compounds of the invention may be prepared as outlined in Scheme Ia. Coupling of an alkyne of formula 1a (Calverley, M. J. and Bretting, C. Aa. S.; Bioorg. Med. Chem. Lett. 9 1841-1844 1993) to an arylic side chain fragment of formula II in the presence of catalytic amounts of a palladium complex such as Pd(PPh3)4 or Pd(PPh3)2Cl2, catalytic amounts of copper (I) iodide and an organic amine base as solvent, gives an intermediate of formula IIIa. The 22-triple bond in IIIa is reduced to a 22- cis-double bond with hydrogen in the presence of catalytic amounts of a Lindlar catalyst.
The conversion of the intermediate IVa to Ia involves a photoisomerisation step and a deprotection step, analogous to the steps used in last stages of the synthesis of other vitamin D analogues, cf. EP patent No. 0 227 826.
The compounds of formula Ib (20-epimers of Ia) are prepared analogously with the 20-epimer 1b (Bretting C, Mxc3x8rk Hansen C, Rastrup Andersen N 1994 Chemistry and biologi of 22,23-yne analogs of calcitriol. In: Norman A W, Bouillon R, Thomasset M (eds.) Vitamin D xe2x80x94A Pluripotent Steroid Hormone: Structural Studies, Molecular Endocrinology and Clinical Applications. Walter de Gruyter, Berlin-N.Y., pp.73-74) as starting material as outlined in Scheme Ib.
The side chain building blocks of formula II are either known compounds or may be prepared as outlined in Scheme II.
Alternatively the compounds of the invention may be prepared as outlined in Scheme III. A vitamin D analogue of formula 2b (Calverley M. J., Tetrahedron, 43, 4609, (1987)) with an aldehyde carbonyl in position 22 reacts with an alkylidene phosphorane of formula X to give a mixture of a 22(Z) and a 22(E) isomers from which the 22(Z) isomer IV is isolated by chromatography. 
Notes to scheme Ia and Ib:
X1 is bromine or iodine
Y is hydrogen, hydroxy or a protected hydroxy group
X, R1, R2 and R3 are as defined above.
a) Heck coupling with Pd(PPh3)2Cl 2/copper(I) iodide as catalyst.
b) Hydrogenation in the presence of a Lindlar catalyst.
c) Isomerisation with hv in the presence of a triplet sensitizer, e.g. anthracene.
d) Deprotection with TBAF or HF. 
The following standard abbreviations are used throughout this disclosure: But=tert-butyl, DMF=N,N-dimethylformamide, DMAP=4-dimethylaminopyridine Et=ethyl, Ether=diethyl ether, pet.ether=petroleum ether, Me=methyl, PPTS=pyridinium p-toluenesulfonate, Py=pyridine, TBAF=tetra-n-butylammonium fluoride, TBS=tert-butyldimethylsilyl, THF=tetrahydrofuran, THP=tetrahydro-4H-pyran-2-yl, Ts=tosyl.
In order to demonstrate the effectiveness of the compounds of formula I an assay for the rating of test compounds for antiproliferative activity in skin cells, e.g. antipsoriatic effect, such as the in vitro assay using HaCaT, a spontaneously immortalized, non-tumorigenic human skin keratinocyte cell line (Mxc3x8rk Hansen, C. et al., J. Invest. Dermatol. 1, 44-48 (1996)), measuring 3H-thymidine uptake can be used.
Generally, the classical effects of 1,25(OH)2D3 on the calcium balance in the organism, including calcemic and calciuric activities, are unwanted in the vitamin D analogues of the present invention, in which selectivity for e.g. inhibition of the proliferation of certain cells, absence of calcemic effects and skin irritation is desired. Thus, the calcemic activity of the compounds may be determined in rats in vivo, as previously described (Binderup, L., Bramm, E., Biochem. Pharmacol. 37, 889-895 (1988)).
Furthermore, the binding to the vitamin D receptor relative to the binding of Calcitriol of the present compounds compared to compounds of the prior art may be determined in vitro as previously described (Binderup, L., Bramm, E., Biochem. Pharmacol. 37, 889-895 (1988)).
The present compounds are intended for use in pharmaceutical compositions which are useful in the local or sytemic treatment of human and veterinary disorders such as cancer, leukemia, myelofibrosis, and psoriasis, of a number of disease states including hyperparathyroidism, particularly secondary hyperparathyroidism associated with renal failure, diabetes mellitus, hypertension, acne, alopecia, skin ageing, AIDS, neurodegenerative disorders such as Alzheimer""s disease, host versus graft reactions, rejection of transplants, for prevention and/or treatment of steroid induced skin atrophy, and for promoting osteogenesis and treating osteoporosis.
The present compounds may be used in combination with other pharmaceuticals or treatment modalities. In the treatment of psoriasis the present compounds may be used in combination with other antipsoriatic drugs, e.g. steroids, or with other treatments e.g. light- or UV-light-treatment or the combined PUVA-treatment. In the treatment of cancer the present compounds may be used in combination with other anti-cancer drugs or anti-cancer treatments, such as radiation treatment. In the prevention of graft rejection and graft versus host reaction, or in the treatment of auto-immune diseases, the present compounds may advantageously be used in combination with other immunosuppressive/immunoregulating drugs or treatments, e.g. with cyclosporin A.
The amount required of a compound of formula I (hereinafter referred to as the active ingredient) for therapeutic effect will, of course, vary both with the particular compound, the route of administration and the mammal under treatment. The compounds of the invention can be administered by the parenteral, intra-articular, enteral or topical routes. They are well absorbed when given enterally and this is the preferred route of administration in the treatment of systemic disorders. In the treatment of dermatological disorders like psoriasis or eye diseases topical or enteral forms are preferred.
While it is possible for an active ingredient to be administered alone as the raw chemical, it is preferable to present it as a pharmaceutical formulation. Conveniently, the active ingredient comprises from 0.1 ppm to 0.1% by weight of the formulation.
The formulations, both for veterinary and for human medical use, of the present invention thus comprise an active ingredient in association with a pharmaceutically acceptable carrier therefore and optionally other therapeutic ingredient(s). The carrier(s) must be xe2x80x9cacceptablexe2x80x9d in the sense of being compatible with the other ingredients of the formulations and not deleterious to the recipient thereof.
The formulations include e.g. those in a form suitable for oral, ophthalmic, rectal, parenteral (including subcutaneous, intramuscular and intravenous), transdermal, intra-articular and topical administration.
By the term xe2x80x9cdosage unitxe2x80x9d is meant a unitary, i.e. a single dose which is capable of being administered to a patient, and which may be readily handled and packed, remaining as a physically and chemically stable unit dose comprising either the active material as such or a mixture of it with solid or liquid pharmaceutical diluents or carriers.
The formulations may conveniently be presented in dosage unit form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation.
Formulations of the present invention suitable for oral administration may be in the form of discrete units as capsules, sachets, tablets or lozenges, each containing a predetermined amount of the active ingredient; in the form of a powder or granules; in the form of a solution or a suspension in an aqueous liquid or non-aqueous liquid; or in the form of an oil-in-water emulsion or a water-in-oil emulsion. The active ingredient may also be administered in the form of a bolus, electuary or paste.
Formulations for rectal administration may be in the form of a suppository incorporating the active ingredient and a carrier, or in the form of an enema.
Formulations suitable for parenteral administration conveniently comprise a sterile oily or aqueous preparation of the active ingredient which is preferably isotonic with the blood of the recipient. Transdermal formulations may be in the form of a plaster or a patch.
Formulations suitable for intra-articular or ophthalmic administration may be in the form of a sterile aqueous preparation of the active ingredient which may be in microcrystalline form, e.g. in the form of an aqueous microcrystalline suspension. Liposomal formulations or biodegradable polymer systems may also be used to present the active ingredient for both intra-articular and ophthalmic administration.
Formulations suitable for topical or ophthalmic administration include liquid or semi-liquid preparations such as liniments, lotions, gels, applicants, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes; or solutions or suspensions such as drops.
In addition to the aforementioned ingredients, the formulations of this invention may include one or more additional ingredients, such as diluents, binders, preservatives etc.
The compositions may further contain other therapeutically active compounds usually applied in the treatment of the above mentioned pathological conditions, such as other immunosuppressants in the treatment of immunological diseases, or steroids in the treatment of dermatological diseases.
The present invention further concerns a method for treating patients suffering from one of the above pathological conditions, said method consisting of administering to a patient in need of treatment an effective amount of one or more compounds of formula I, alone or in combination with one or more excipients or other therapeutically active compounds usually applied in the treatment of said pathological conditions. The treatment with the present compounds and/or with further therapeutically active compounds may be simultaneous or with intervals.
In the systemic treatment daily doses of from 0.001-2 xcexcg per kilogram bodyweight, preferably from 0.002-0.3 xcexcg/kg of mammal bodyweight, for example 0.003-0.3 xcexcg/kg of a compound of formula I are administered, typically corresponding to a daily dose for an adult human of from 0.2 to 25 xcexcg. In the topical treatment of dermatological disorders, ointments, creams or lotions containing from 0.1-1000 xcexcg/g, and preferably from 1-500 xcexcg/g, and more preferably from 10-250 xcexcg/g, of a compound of formula I are administered. For topical use in ophthalmology ointments, drops or gels containing from 0.1-1000 xcexcg/g, and preferably from 1-500 xcexcg/g, more preferably from 10-250 xcexcg/g, of a compound of formula I are administered. The oral compositions are formulated, preferably as tablets, capsules, or drops, containing from 0.05-100 xcexcg, preferably from 0.1-50 xcexcg, of a compound of formula I, per dosage unit.
The invention will now be further described in the following General Procedures, Preparations and Examples